Original Communication - Detection of two rare β-thalassemia mutations [-90 (C → T) and CD 26 (C →T)] among Indians

BACKGROUND: β -Thalassemia (β-thal) is present in practically every caste group in Indians. Molecular characterization of β-thal in these groups has revealed an extremely heterogeneous picture. AIM: To identify all the mutations and to detect the novel mutations using a versatile mutation detection technique. MATERIALS AND METHODS: Denaturing gradient gel electrophoresis (DGGE) has been established to scan the entire β-globin gene to localize the mutation followed by DNA sequencing for characterization. The DNA samples from two families referred to us either for prenatal diagnosis or for DNA studies were studied. RESULTS: Atypical DGGE patterns in fragments B & A indicating the presence of the mutation, have been detected in both the families. DNA sequencing revealed two rare patterns fragments with patterns in fragments β-thal mutations [CD 26 (C→T) and -90 (C →T)]. CONCLUSION: DGGE is a useful mutation detection technique to identify β-thal mutations among the heterogeneous Indian population

Original Communication - Detection of two rare β-thalassemia mutations [-90 (C → T) and CD 26 (C →T)] among Indians

BACKGROUND: β -Thalassemia (β-thal) is present in practically every caste group in Indians. Molecular characterization of β-thal in these groups has revealed an extremely heterogeneous picture. AIM: To identify all the mutations and to detect the novel mutations using a versatile mutation detection technique. MATERIALS AND METHODS: Denaturing gradient gel electrophoresis (DGGE) has been established to scan the entire β-globin gene to localize the mutation followed by DNA sequencing for characterization. The DNA samples from two families referred to us either for prenatal diagnosis or for DNA studies were studied. RESULTS: Atypical DGGE patterns in fragments B & A indicating the presence of the mutation, have been detected in both the families. DNA sequencing revealed two rare patterns fragments with patterns in fragments β-thal mutations [CD 26 (C→T) and -90 (C →T)]. CONCLUSION: DGGE is a useful mutation detection technique to identify β-thal mutations among the heterogeneous Indian population

Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma

Background: Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk. Aim: To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma. Settings and Design: Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study. Materials and Methods: Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation. Results and Conclusions: There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable

Supplementary Material for: Phenotyping and Genotyping of HNA: Prevalence, Risk of Alloimmunization, and HNA Incompatibilities in Indians

Background: Antibodies to human neutrophil alloantigens (HNA) are involved in the pathophysiology of several clinical conditions including transfusion-related acute lung injury (TRALI), alloimmune and autoimmune neutropenia, and febrile nonhemolytic transfusion reactions leading to neutropenia. The cognate antigens are polymorphic structures expressed on several glycoproteins on the neutrophils, i.e., antigens HNA-1a, -1b, -1c, and -1d on Fc-γ-receptor IIIb; HNA-2 on CD177; HNA-3a and -3b on choline transporter-like protein 2; HNA-4a and -4b on CD11b/αM subunit of the αMβ2-integrin (CD11b/CD18, Mac-1, CR3); and HNA-5a and -5b on αL-subunit (CD11a) of the αLβ2 integrin (CD11a/CD18), leukocyte function associated molecule (LFA)-1. Currently, there is a lacuna of diagnostic methods for detection of HNA in India. This study aimed to determine the HNA frequencies in Indians, estimate the risk of alloimmunization, and prepare typed neutrophil panels, which can be used to detect HNA antibodies in neutropenia cases. Material and Methods: EDTA blood samples were collected from random 1,054 blood donors. HNA-2 was phenotyped on fresh EDTA samples using FITC labelled monoclonal anti-CD177 by flowcytometry. HNA-1 (FCGR3B) genotyping was carried out by DNA sequencing and PCR-RFLP. Antigens of HNA-3 (SLC44A2) and HNA-5 (ITGAL) were genotyped by PCR-RFLP using TaqαI and Bsp1286I restriction enzymes, respectively, while HNA-4 (ITGAM) was genotyped by PCR-SSP. Results: Allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were found to be 0.433, 0.444, and 0.087, respectively. FCGR3B*01+*02+*03− was the most common genotype (33.78%). Ten individuals showed deficiency of FCGR3B individuals, while 23 showed hyperexpression, i.e., FCGR3B*01+*02+*03+. FCGR3B*04and *05 occurred with a frequency of 0.002 and 0.024. HNA-2 was found to be a high frequency antigen occurring in 98.8% population. Four percent individuals showed atypical expression of CD177 on their neutrophils. Allele frequencies of SLC44A2*01 and SLC44A2*02were 0.812 and 0.188, respectively, and that of ITGAM*01, ITGAM*02, ITGAL*01, and ITGAL*02 were 0.9546, 0.0454, 0.2372, and 0.7628, respectively. Conclusion: This is the first study in India to report the frequencies of HNA among blood donors. Typed neutrophil panels identified in the present study will enable us to investigate suspected cases of immune neutropenia in future

Analysis of Complement Receptor Type 1 (CR1) Polymorphisms and Its Association With Malaria in Rural Population of Maharashtra

The interaction between human host and the Plasmodium parasite is complex. The factors affecting the causality of infection and its severity are yet not completely understood. Single Nucleotide Polymorphisms (SNP) associated with CR1 may be associated with patho-physiology of malaria and its susceptibility to the disease. Methods: The objective of the present study was to calculate the incidence of various antigens of Knops blood group system and CR1 Exon22 polymorphisms in rural population from Chiplun Taluka of Ratnagiri district. The study included 112 malaria positive cases and 909 healthy controls, which were screened for CR1 Exon22 polymorphism. Knops (Kna/b), McCoy (McCa/b), Swain-Langley (Sl1/2) polymorphisms were screened in 93 cases and 321 healthy controls. The frequencies were determined using a PCR-RFLP technique. Results: Only wild types of the allele form were observed in Knops blood group system in malaria cases and healthy control. CR1 exon22 polymorphism was seen in the study population with all the 3 allele type distributed in the cases and control samples. No significant allelic or genotypic differences were found between the controls and the disease groups. Conclusion: The results of the present study demonstrate that common CR1 Exon22 and Knops blood group system are not associated with malaria in the endemic area